CN109825537A - A kind of bafillus natto liquor fermentation prepares vitamin K2Method - Google Patents
A kind of bafillus natto liquor fermentation prepares vitamin K2Method Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 44
- 230000004151 fermentation Effects 0.000 title claims abstract description 43
- 235000013557 nattō Nutrition 0.000 title claims abstract description 26
- 229930003231 vitamin Natural products 0.000 title description 4
- 239000011782 vitamin Substances 0.000 title description 4
- 229940088594 vitamin Drugs 0.000 title description 4
- 235000013343 vitamin Nutrition 0.000 title description 4
- 150000003722 vitamin derivatives Chemical class 0.000 title description 4
- 239000001963 growth medium Substances 0.000 claims abstract description 87
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000002609 medium Substances 0.000 claims abstract description 17
- 239000002994 raw material Substances 0.000 claims abstract description 11
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- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 6
- PFRQBZFETXBLTP-RCIYGOBDSA-N 2-[(2e,6e,10e,14e,18e)-3,7,11,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaen-1-yl]-3-methyl-1,4-dihydronaphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 PFRQBZFETXBLTP-RCIYGOBDSA-N 0.000 claims abstract 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 40
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 30
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- 239000001888 Peptone Substances 0.000 claims description 25
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- 108010059892 Cellulase Proteins 0.000 claims description 15
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- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 15
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- 238000003756 stirring Methods 0.000 claims description 14
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- 239000008103 glucose Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 8
- 238000004090 dissolution Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- DKHGMERMDICWDU-GHDNBGIDSA-N menaquinone-4 Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 DKHGMERMDICWDU-GHDNBGIDSA-N 0.000 claims description 6
- 108090000787 Subtilisin Proteins 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
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- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 7
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- FRASJONUBLZVQX-UHFFFAOYSA-N naphthoquinone group Chemical group C1(C=CC(C2=CC=CC=C12)=O)=O FRASJONUBLZVQX-UHFFFAOYSA-N 0.000 description 1
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- MBWXNTAXLNYFJB-NKFFZRIASA-N phylloquinone Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 description 1
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Abstract
A kind of method that bafillus natto liquor fermentation prepares farnoquinone, including pretreatment of raw material and culture medium preparations, vacuum cooking processing, the compound enzymatic treatment of high temperature, Protease Treatment, Low Temperature Plasma Treating, except Slag treatment and fermented and cultured.The present invention in conjunction with protease hydrolyzed technology, pre-processes the semisynthetic medium of high solid content in the case where depressurizing conditions of cooking, to form clear liquid, avoids a series of disadvantages of high solid content culture medium using lower temperature plasma technology；Meanwhile by pretreated semisynthetic medium, no longer need to carry out high pressure moist heat sterilization, the viscosity of culture medium is reduced while reducing energy consumption, is conducive to the fermenting and producing in later period and isolates and purifies.
Description
Technical field
The present invention relates to a kind of bafillus natto liquor fermentations to prepare vitamin K2Method, belong to biotechnology neck
Domain.
Background technique
Vitamin K2It is a series of isoprene containing 2- methyl-1,4-naphthaquinone parent nucleus and C3 with number not etc.
The general designation of the terpenes side chain compound of structural unit can be divided into K2 (10), K2 according to the number of carbon on terpenes side chain
(20), K2 (35), K2 (40) etc..Vitamin K2It is a kind of liposoluble vitamin, the naphthoquinones group with phylloquinone bioactivity
Derivative, be one of indispensable important vitamin in human body.With having been demonstrated that its functional research, dimension is given birth to
Plain K2Have the function of the diseases such as pre- preventing bone rarefaction, angiocarpy, Parkinson, liver cancer.Therefore, functional food, medical products, change
The fields such as cosmetic are to vitamin K2Demand increasingly increase, good market prospect.
Vitamin K2Acquisition methods it is usual there are three types of: (1) extract and obtain from natural organism；(2) in height
It is obtained under warm hyperbaric environment using the method for chemical industry synthesis；(3) it is controlled in the culture substrate of artificial or semi-artificial synthesis certain
Condition of culture utilizes micro-organisms vitamin K2.There is yields not high, with high costs, seriously polluted etc. to ask for first two method
Topic produces vitamin K using microbial fermentation in contrast2It is a kind of safe and efficient, environmental-friendly new methods.Mesh
Before, microbial fermentation produces vitamin K2Method, be primarily present following two technological difficulties: (1) efficiently synthesizing vitamin K2's
The acquisition of bacterial strain, (2) control good condition of culture in order to obtain the vitamin K of high concentration2Fermentation liquid.
Bafillus natto is a kind of direct-edible probiotics as a subspecies in bacillus subtilis.Not
The natural bacterium colony of engineered mistake inherently vitamin K with higher2Synthesis capability and the daily acquisition vitamin K of people2's
A kind of main path.Vitamin K is synthesized using bacillus natto to ferment2Except using expensive artificial synthetic medium
Outside, semisynthetic medium as main component can also be made using eutrophic agricultural and sideline products such as bean powder, bean dregs and grain dusts.But
It is that there is no this kind of semisynthetic medium high yield vitamin K of effective use at present2Method.Its Major Difficulties is bean powder, bean dregs
And the agricultural and sideline products such as corn flour are insoluble ingredients, during the fermentation, these substances will lead to the biography in fermentor system
Oxygen quotient reduces, and oxygen is difficult to good distribution in fermentation tank, causes the growth of thallus and anabolism to be affected, together
When due to the solid content in medium component it is higher (usually more than 20%), to guarantee enough mixabilities, it has to mention
The revolving speed of agitating paddle in high fermentor, this results in the significant increase of energy consumption in incubation again.Further, since purebred culture
It needs, the necessary high-temperature sterilization of all the components before fermentation, the agricultural and sideline products such as usual bean powder will lead to training after high-temperature sterilization
The raising of base viscosity is supported, this further increases the energy consumption of incubation.
At present existing research shows that simply substituting the agricultural and sideline productions such as bean powder, bean dregs, the corn flour in semisynthetic medium
Product ingredient is difficult to ensure the growth conditions of bafillus natto, thus utilize not yet with semisynthetic medium culture be mainly at
The bafillus natto divided efficiently synthesizes vitamin K2Method.
Summary of the invention
The purpose of the present invention is to provide a kind of bafillus natto liquor fermentations to prepare vitamin K2Method.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that a kind of bafillus natto
Liquor fermentation prepares vitamin K2Method, including the following steps:
Step 1: by mixed raw material powder, peptone, sodium chloride, K2HPO4、MgSO4With water be uniformly mixed, make mixing after material it is exhausted
It is 5 ~ 8g/L to nitrogen content, peptone concentration is 30 ~ 100g/L, sodium chloride concentration 1.8-2.3g/L, K2HPO4Concentration is 1.2-
2.0g/L、MgSO4Concentration is 0.3-0.6g/L, obtains culture medium, spare；
Step 2: the culture medium being placed in a cooker, is then vacuumized, then maintains the air pressure in the cooker
It is heated 15 ~ 25 minutes in -0.5 ~ -0.3Mpa, and under the conditions of 80 ~ 90 DEG C, during heating with the revolving speed of 100 ~ 250rpm
Culture medium is stirred；
Step 3: the pH value of the culture medium in adjustment cooker to 6.0 ~ 7.0 is added Thermostable α-Amylase solution, maintains step
2 temperature, adjusting the air pressure in cooker is -0.2 ~ -0.05MPa, is handled 30-60 minutes；Next temperature in adjustment cooker
Degree is 45-55 DEG C, and pH value is 5.0 ~ 6.0, addition cellulase solution and xylanase solution, stir process 30-60 minutes；
Step 4: Protease Treatment
Step 3 culture medium obtained is cooled to 30-40 DEG C, the fibrinoclase that every liter of culture medium adds 1 ~ 6ml is molten
Liquid, adjusting pH is 6.0 ~ 7.0, with speed of agitator 450rpm-1000rpm, is stirred 30 ~ 90 minutes；
Step 5: the temperature in boiling vessel being maintained 25 ~ 40 DEG C, is that 100 ~ 250rpm is stirred with revolving speed, is purged with nitrogen
The cavity of boiling vessel, nitrogen flow rate 0.1-1L/min continue 5-10min, then by the culture medium in boiling vessel with 0.2-2
Culture medium of the L/min flow velocity by Low Temperature Plasma Treating equipment, after obtaining Low Temperature Plasma Treating；
Step 6: the clear liquid obtained after the culture medium after Low Temperature Plasma Treating that step 5 obtains is filtered with plate filter
As fermentation medium；
Step 7: fermentation prepares vitamin K2, including the following steps:
S1, by after the bacterium liquid activation saved in glycerol tube or inclined-plane, be inoculated in culture medium A, culture obtain the first culture solution, so
The first culture solution is inoculated in culture medium B afterwards, culture obtains the second culture solution, and the second culture solution is then inoculated in culture medium
In C, culture obtains high vigor kind liquid；The culture medium A: glucose 8-12g/L, peptone 8-12g/L, NaCl4-6g/L are used
After water dissolution, adjustment pH value is 7.0 ~ 7.4；Culture medium B: glucose 3-5g/l, peptone 12-18g/l, NaCl 3-5g/l,
K2HPO40.8-1.2g/l, after being dissolved with the clear liquid that step 6 obtains, adjustment pH value is 6.5 ~ 7.0；Culture medium C: peptone 25-
35g/l, NaCl 1.5-2.5g/l, K2HPO41-2g/l, MgSO40.4-0.6g/l is dissolved with the clear liquid obtained with step 6；
S2, by S1 step to high vigor kind liquid be seeded to the fermentation medium and cultivate.
Preferred technical solution are as follows: the enzyme activity of Thermostable α-Amylase solution is greater than or equal to 2000U/ml, every liter of training
It supports base and adds 1 ~ 6ml Thermostable α-Amylase solution.
Preferred technical solution are as follows: the enzyme activity of the xylanase solution is greater than or equal to 2000U/ml, every liter of culture
Base adds 1 ~ 6ml xylanase solution.
Preferred technical solution are as follows: the enzyme activity of the cellulase solution is greater than or equal to 2000U/ml, every liter of culture
Base adds 1 ~ 6ml cellulase solution.
Preferred technical solution are as follows: the fibrinoclase of fibrinoclase solution is subtilisin, natto
The enzyme activity of at least one of kinases, serine protease, fibrinoclase solution is greater than or equal to 2000U/ml.
Preferred technical solution are as follows: the pressure when plate filter filters is 0.2 ~ 0.5MPa, and pore size filter is
0.8um, flow are 0.5 ~ 8m3/h。
Preferred technical solution are as follows: it is 250 ~ 750rpm, ventilation that revolving speed is kept stirring during the fermented and cultured of S2 step
Amount is 1 ~ 5vvm.
Preferred technical solution are as follows: the relative energy density of the Low Temperature Plasma Treating equipment is 25 ~ 60kW/l, electricity
Polar plate interval is 4-10mm, electrode diameter 40-120mm.
Since above-mentioned technical proposal is used, the present invention has the advantage, that compared with prior art
The present invention uses lower temperature plasma technology in conjunction with protease hydrolyzed technology, in the case where depressurizing conditions of cooking to high solid content
The semisynthetic medium of content is pre-processed, and to form clear liquid, avoids a series of disadvantages of high solid content culture medium；Together
When, by pretreated semisynthetic medium, no longer needs to carry out high pressure moist heat sterilization, reduce culture medium while reducing energy consumption
Viscosity, be conducive to the fermenting and producing in later period and isolate and purify.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this implementation
Content disclosed by example is understood other advantages and efficacy of the present invention easily.
The purpose of term used herein, which is only that, illustrates particular embodiment, it is not intended to be limited to the present invention.It removes
Non- context is explicitly shown, otherwise singular " one " used herein, "one" also include plural form.
When illustrating preferred embodiment, it is potentially based on clear purpose and uses special term；However, this specification institute
Revealer is not intended to be limited in the selected special term；And it is to be understood that each particular element includes having
Identical function, all equivalence techniques for operating in a similar manner and reaching similar effects.
Embodiment 1: a kind of bafillus natto liquor fermentation prepares vitamin K2Method
A kind of bafillus natto liquor fermentation prepares vitamin K2Method, including following technology contents.
(1) pretreatment of raw material and culture medium are prepared
The raw material is one of soya bean, mung bean, the corn and soybean dregs of rice, wheat bran, stalk or a variety of.Different material is distinguished
It is dry to be lower than 10% to water content, 80 mesh are then crushed to, it is spare.The present invention is specifically chosen Soybean Meal.
Prepare mixed raw material powder.Drying, smashed different material are mixed in proportion, make mixing after material it is exhausted
It is 6.5%(w% to nitrogen content), sugared content 40% obtains mixed raw material powder, spare.
Culture medium is prepared.By mixed raw material powder, peptone, sodium chloride, K2HPO4、MgSO4, fermentation use water, by a certain percentage
Mixing, make mixing after material absolute nitrogen content 6.5g/l(N%), peptone concentration 65g/L, sodium chloride concentration 2g/L,
K2HPO4Concentration is 1.6g/L, MgSO4Concentration is 0.45g/L, obtains culture medium, spare.
(2) vacuum cooking is handled
The volume of cooker is different, selects suitable vacuum pump, air pressure in cooker is extracted out, maintains -0.4MPa, and stirring turns
Speed is 180rpm.Cooker is heated to boiling using jacket water (J.W.), temperature is 85 DEG C at this time, is maintained 20 minutes.
(3) the compound enzymatic treatment of high temperature
Adjust cooker in pH be 6.5, be added Thermostable α-Amylase, maintain stirring and temperature, adjust cooker in air pressure be-
0.1MPa is handled 45 minutes.Adjusting temperature in cooker is 50 DEG C, pH 5.5, and cellulase and zytase is added, and is maintained
Stirring and air pressure are handled 45 minutes.
Amylase solution vigor should be not less than 2000U/ml, and every liter of cooking liquor volume adds 3.5ml amylase solution.
Xylanase solution vigor should be not less than 2000U/ml, and every liter of cooking liquor volume adds 3.5ml xylanase solution.
Cellulase solution vigor should be not less than 2000U/ml, and every liter of cooking liquor volume adds 3.5ml cellulase solution.
(4) Protease Treatment
Beans and its product for high protein content, the celloglobulin being rich in have passed through abovementioned steps and have broken eucaryotic cell structure
It badly discharges, is conducive to fibrinous fracture in high shear environment.Step (3) raw material obtained is cooled down in boiling vessel
To 37 DEG C, every liter of volume addition 3.5ml adds fibrinoclase solution, and adjusting pH is 6.5, improves speed of agitator extremely
700rpm is maintained 60 minutes.
Fibrinoclase includes subtilisin, and Nattokinase, serine protease etc., vigor is not lower than
2000U/ml。
Agitating paddle form in boiling vessel should be high shear paddle type, such as six flat leaf paddles；It is 1/4 ~ 1/ with boiling vessel diameter ratio
3。
(5) Low Temperature Plasma Treating
Electronics that low-temperature plasma is formed by electrion, ion, neutral ion can play the role of sterilizing.Low temperature etc. from
Daughter exists simultaneously perforation and electrostatic interference effect, can destroy the eucaryotic cell structure in pretreatment fluid, release and wherein include
A variety of large biological molecules.And etching and Free Radical can destroy certain chemical bonds, destroy the advanced knot of large biological molecule
Structure, causes the fracture of DNA and part long-chain macromolecule, and the insoluble nutriment in part can release.
It is 30 DEG C that temperature is maintained in boiling vessel, revolving speed 170rpm.With N2Cavity is swept in air-blowing, and flow velocity is 0.5 L/min, is held
Continuous 8min.The culture medium that step (4) are obtained passes through Low Temperature Plasma Treating equipment, relative energy density with 1L/min flow velocity
(specific energy density, SED) is 40kW/l, and 7mm, electrode diameter 80mm are divided between electrode plate, obtains low temperature etc.
Gas ions treated culture medium.
(6) Slag treatment is removed
Culture medium after step (5) processing is handled by plate filter.Filter pressure is 0.35MPa, and pore size filter is
0.8um, flow 0.65m3/ h obtains the stillness of night and filter residue after filtering.
(7) seed culture
Seed culture medium includes the different culture medium of three classes.It is specific as follows: culture medium A: glucose 10g/l, peptone 10g/l,
Adjustment pH is 7.2 after NaCl 5g/l is dissolved with water；Culture medium B: glucose 5g/l, peptone 15g/l, NaCl 4g/l,
K2HPO4Adjustment pH is 6.7 after the clear liquid dissolution that 1g/l step (6) obtains；Culture medium C: peptone 30g/l, NaCl 2g/l,
K2HPO41.5g/l, MgSO4The clear liquid dissolution that 0.5g/l step (6) obtains.
Seed culture process includes gradually passing on.By the bafillus natto bacterium solution saved in glycerol tube or inclined-plane according to skill
Art require activation after, be inoculated in culture medium A with 10% inoculum concentration, 150rpm stirring, 37 DEG C culture for 24 hours, by the culture solution with
10% inoculum concentration is inoculated in culture medium B, which for 24 hours, is inoculated in training with 10% inoculum concentration by 200rpm stirring, 37 DEG C of cultures
It supports in base C, 200rpm stirring, 37 DEG C of culture 12h obtain high vigor kind liquid.
(8) fermented and cultured
High vigor kind liquid is inoculated in the clear liquid of step (6) acquisition, is cultivated in fermentor, is kept during fermented and cultured
Speed of agitator is 500rpm, ventilatory capacity 3vvm.Bafillus natto is aerobic bacteria, keeps enough oxygen supplies effective
Improve the growth of thallus.Feed supplement can be carried out according to the variation of the nitrogen content of detection in fermentation process, to extend fermentation time.
When residual sugar is lower than 0.5g/l in fermentation liquid, fermentation can be terminated, obtains and is higher than 100mg/L vitamin K2Concentration
Fermentation liquid.
Embodiment 2: a kind of bafillus natto liquor fermentation prepares vitamin K2Method
A kind of bafillus natto liquor fermentation prepares vitamin K2Method, including the following steps:
Step 1: by mixed raw material powder, peptone, sodium chloride, K2HPO4、MgSO4With water be uniformly mixed, make mixing after material it is exhausted
It is 5g/L to nitrogen content, peptone concentration 30g/L, sodium chloride concentration 1.8g/L, K2HPO4Concentration is 1.2g/L, MgSO4It is dense
Degree is 0.3g/L, obtains culture medium, spare；
Step 2: the culture medium being placed in a cooker, is then vacuumized, then maintains the air pressure in the cooker
It heats 15 minutes in -0.5Mpa, and under the conditions of 80 DEG C, culture medium is stirred with the revolving speed of 100rpm during heating
It mixes；
Step 3: the pH value of the culture medium in adjustment cooker to 6.0 is added Thermostable α-Amylase solution, maintains step 2
Temperature, adjusting the air pressure in cooker is -0.2MPa, is handled 30 minutes；Next temperature is 45 DEG C in adjustment cooker, pH value
It is 5.0, addition cellulase solution and xylanase solution, stir process 30 minutes；
Step 4: Protease Treatment
Step 3 culture medium obtained is cooled to 30 DEG C, the fibrinoclase solution of every liter of culture medium addition 1ml is adjusted
Saving pH is 6.0, with speed of agitator 450rpmrpm, is stirred 30 minutes；
Step 5: the temperature in boiling vessel being maintained 25 DEG C, is stirred with revolving speed for 100rpm, boiling vessel is purged with nitrogen
Cavity, nitrogen flow rate 0.1L/min, continue 5min, then the culture medium in boiling vessel is passed through with 0.2 L/min flow velocity
Low Temperature Plasma Treating equipment, the culture medium after obtaining Low Temperature Plasma Treating；
Step 6: the clear liquid obtained after the culture medium after Low Temperature Plasma Treating that step 5 obtains is filtered with plate filter
As fermentation medium；
Step 7: fermentation prepares vitamin K2, including the following steps:
S1, by after the bacterium liquid activation saved in glycerol tube or inclined-plane, be inoculated in culture medium A, culture obtain the first culture solution, so
The first culture solution is inoculated in culture medium B afterwards, culture obtains the second culture solution, and the second culture solution is then inoculated in culture medium
In C, culture obtains high vigor kind liquid；The culture medium A: glucose 8g/L, peptone 8g/L, NaCl4g/L, after being dissolved with water,
Adjusting pH value is 7.0；Culture medium B: glucose 3g/l, peptone 12g/l, NaCl 3g/l, K2HPO40.8g/l is obtained with step 6
After the clear liquid dissolution obtained, adjustment pH value is 6.5；Culture medium C: peptone 25g/l, NaCl 1.5g/l, K2HPO41g/l,
MgSO40.4g/l is dissolved with the clear liquid obtained with step 6；
S2, by S1 step to high vigor kind liquid be seeded to the fermentation medium and cultivate.
Preferred embodiment are as follows: the enzyme activity of Thermostable α-Amylase solution is 3000U/ml, every liter of culture medium addition
1ml Thermostable α-Amylase solution.
Preferred embodiment are as follows: the enzyme activity of the xylanase solution is 3500U/ml, and every liter of culture medium adds 1ml
Xylanase solution.
Preferred embodiment are as follows: the enzyme activity of the cellulase solution is 3200U/ml, and every liter of culture medium adds 1ml
Cellulase solution.
Preferred embodiment are as follows: the fibrinoclase of fibrinoclase solution is subtilisin, fiber
The enzyme activity of protein dissolution enzyme solutions is 2000U/ml.
Preferred embodiment are as follows: the plate filter filter when pressure be 0.2MPa, pore size filter 0.8um,
Flow is 0.5m3/h。
Preferred embodiment are as follows: it is 250rpm that revolving speed is kept stirring during the fermented and cultured of S2 step, and ventilatory capacity is
1vvm。
Preferred embodiment are as follows: the relative energy density of the Low Temperature Plasma Treating equipment is 25kW/l, electrode
4mm, electrode diameter 40mm are divided between plate.
Embodiment 3: a kind of bafillus natto liquor fermentation prepares vitamin K2Method
A kind of bafillus natto liquor fermentation prepares vitamin K2Method, including the following steps:
Step 1: by mixed raw material powder, peptone, sodium chloride, K2HPO4、MgSO4With water be uniformly mixed, make mixing after material it is exhausted
It is 8g/L to nitrogen content, peptone concentration 100g/L, sodium chloride concentration 2.3g/L, K2HPO4Concentration is 2.0g/L, MgSO4
Concentration is 0.6g/L, obtains culture medium, spare；
Step 2: the culture medium being placed in a cooker, is then vacuumized, then maintains the air pressure in the cooker
It heats 25 minutes in -0.3Mpa, and under the conditions of 90 DEG C, culture medium is stirred with the revolving speed of 250rpm during heating
It mixes；
Step 3: the pH value of the culture medium in adjustment cooker to 7.0 is added Thermostable α-Amylase solution, maintains step 2
Temperature, adjusting the air pressure in cooker is -0.05MPa, is handled 60 minutes；Next temperature is 55 DEG C in adjustment cooker, pH
Value is 6.0, addition cellulase solution and xylanase solution, stir process 60 minutes；
Step 4: Protease Treatment
Step 3 culture medium obtained is cooled to 40 DEG C, the fibrinoclase solution of every liter of culture medium addition 6ml is adjusted
Saving pH is 7.0, with speed of agitator 1000rpm, is stirred 90 minutes；
Step 5: the temperature in boiling vessel being maintained 40 DEG C, is stirred with revolving speed for 250rpm, boiling vessel is purged with nitrogen
Cavity, nitrogen flow rate 1L/min, continue 10min, the culture medium in boiling vessel is then passed through into low temperature with 2 L/min flow velocitys
Apparatus for processing plasma, the culture medium after obtaining Low Temperature Plasma Treating；
Step 6: the clear liquid obtained after the culture medium after Low Temperature Plasma Treating that step 5 obtains is filtered with plate filter
As fermentation medium；
Step 7: fermentation prepares vitamin K2, including the following steps:
S1, by after the bacterium liquid activation saved in glycerol tube or inclined-plane, be inoculated in culture medium A, culture obtain the first culture solution, so
The first culture solution is inoculated in culture medium B afterwards, culture obtains the second culture solution, and the second culture solution is then inoculated in culture medium
In C, culture obtains high vigor kind liquid；The culture medium A: glucose 12g/L, peptone 12g/L, NaCl4-6g/L, use are water-soluble
Xie Hou, adjustment pH value are 7.4；Culture medium B: glucose 5g/l, peptone 18g/l, NaCl 5g/l, K2HPO41.2g/l, with step
After the rapid 6 clear liquid dissolutions obtained, adjustment pH value is 7.0；Culture medium C: peptone 35g/l, NaCl2.5g/l, K2HPO42g/l,
MgSO40.6g/l is dissolved with the clear liquid obtained with step 6；
S2, by S1 step to high vigor kind liquid be seeded to the fermentation medium and cultivate.
Preferred embodiment are as follows: the enzyme activity of Thermostable α-Amylase solution is 5000U/ml, every liter of culture medium addition
6ml Thermostable α-Amylase solution.
Preferred embodiment are as follows: the enzyme activity of the xylanase solution is 5000U/ml, and every liter of culture medium adds 6ml
Xylanase solution.
Preferred embodiment are as follows: the enzyme activity of the cellulase solution is 5000U/ml, and every liter of culture medium adds 6ml
Cellulase solution.
Preferred embodiment are as follows: the fibrinoclase of fibrinoclase solution is subtilisin, natto
Kinases, the mixture constituted according to the mass ratio of 1:1, the enzyme activity of fibrinoclase solution are 5000U/ml.
Preferred embodiment are as follows: the plate filter filter when pressure be 0.5MPa, pore size filter 0.8um,
Flow is 8m3/h。
Preferred embodiment are as follows: it is 750rpm that revolving speed is kept stirring during the fermented and cultured of S2 step, and ventilatory capacity is
5vvm。
Preferred embodiment are as follows: the relative energy density of the Low Temperature Plasma Treating equipment is 60kW/l, electrode
10mm, electrode diameter 120mm are divided between plate.
As described above is only to be not intended to tool to explain the preferred embodiments of the invention to do any shape to the present invention
Limitation in formula should all wrap therefore all have any modification or change for making the related present invention under identical spirit
It includes in the scope that the invention is intended to protect.
Claims (8)
1. a kind of bafillus natto liquor fermentation prepares vitamin K2Method, it is characterised in that: include the following steps:
Step 1: by mixed raw material powder, peptone, sodium chloride, K2HPO4、MgSO4With water be uniformly mixed, make mixing after material it is exhausted
It is 5 ~ 8g/L to nitrogen content, peptone concentration is 30 ~ 100g/L, sodium chloride concentration 1.8-2.3g/L, K2HPO4Concentration is 1.2-
2.0g/L、MgSO4Concentration is 0.3-0.6g/L, obtains culture medium, spare；
Step 2: the culture medium being placed in a cooker, is then vacuumized, then maintains the air pressure in the cooker
It is heated 15 ~ 25 minutes in -0.5 ~ -0.3Mpa, and under the conditions of 80 ~ 90 DEG C, during heating with the revolving speed of 100 ~ 250rpm
Culture medium is stirred；
Step 3: the pH value of the culture medium in adjustment cooker to 6.0 ~ 7.0 is added Thermostable α-Amylase solution, maintains step
2 temperature, adjusting the air pressure in cooker is -0.2 ~ -0.05MPa, is handled 30-60 minutes；Next temperature in adjustment cooker
Degree is 45-55 DEG C, and pH value is 5.0 ~ 6.0, addition cellulase solution and xylanase solution, stir process 30-60 minutes；
Step 4: Protease Treatment
Step 3 culture medium obtained is cooled to 30-40 DEG C, the fibrinoclase that every liter of culture medium adds 1 ~ 6ml is molten
Liquid, adjusting pH is 6.0 ~ 7.0, with speed of agitator 450rpm-1000rpm, is stirred 30 ~ 90 minutes；
Step 5: the temperature in boiling vessel being maintained 25 ~ 40 DEG C, is that 100 ~ 250rpm is stirred with revolving speed, is purged with nitrogen
The cavity of boiling vessel, nitrogen flow rate 0.1-1L/min continue 5-10min, then by the culture medium in boiling vessel with 0.2-2
Culture medium of the L/min flow velocity by Low Temperature Plasma Treating equipment, after obtaining Low Temperature Plasma Treating；
Step 6: the clear liquid obtained after the culture medium after Low Temperature Plasma Treating that step 5 obtains is filtered with plate filter
As fermentation medium；
Step 7: fermentation prepares vitamin K2, including the following steps:
S1, by after the bacterium liquid activation saved in glycerol tube or inclined-plane, be inoculated in culture medium A, culture obtain the first culture solution, so
The first culture solution is inoculated in culture medium B afterwards, culture obtains the second culture solution, and the second culture solution is then inoculated in culture medium
In C, culture obtains high vigor kind liquid；The culture medium A: glucose 8-12g/L, peptone 8-12g/L, NaCl4-6g/L are used
After water dissolution, adjustment pH value is 7.0 ~ 7.4；Culture medium B: glucose 3-5g/l, peptone 12-18g/l, NaCl 3-5g/l,
K2HPO40.8-1.2g/l, after being dissolved with the clear liquid that step 6 obtains, adjustment pH value is 6.5 ~ 7.0；Culture medium C: peptone 25-
35g/l, NaCl 1.5-2.5g/l, K2HPO41-2g/l, MgSO40.4-0.6g/l is dissolved with the clear liquid obtained with step 6；
S2, by S1 step to high vigor kind liquid be seeded to the fermentation medium and cultivate.
2. the method that bafillus natto liquor fermentation according to claim 1 prepares farnoquinone, it is characterised in that: resistance to
The enzyme activity of high-temperatureα-amylase solution is greater than or equal to 2000U/ml, and every liter of culture medium adds 1 ~ 6ml Thermostable α-Amylase
Solution.
3. the method that bafillus natto liquor fermentation according to claim 1 prepares farnoquinone, it is characterised in that: institute
The enzyme activity for stating xylanase solution is greater than or equal to 2000U/ml, and every liter of culture medium adds 1 ~ 6ml xylanase solution.
4. the method that bafillus natto liquor fermentation according to claim 1 prepares farnoquinone, it is characterised in that: institute
The enzyme activity for stating cellulase solution is greater than or equal to 2000U/ml, and every liter of culture medium adds 1 ~ 6ml cellulase solution.
5. bafillus natto liquor fermentation according to claim 1 prepares vitamin K2Method, it is characterised in that: it is fine
Fibrillarin dissolves the fibrinoclase of enzyme solutions at least one in subtilisin, Nattokinase, serine protease
Kind, the enzyme activity of fibrinoclase solution is greater than or equal to 2000U/ml.
6. bafillus natto liquor fermentation according to claim 1 prepares vitamin K2Method, it is characterised in that: institute
Pressure when stating plate filter filtering is 0.2 ~ 0.5MPa, and pore size filter 0.8um, flow is 0.5 ~ 8m3/h。
7. bafillus natto liquor fermentation according to claim 1 prepares vitamin K2Method, it is characterised in that: S2
It is 250 ~ 750rpm that revolving speed is kept stirring during the fermented and cultured of step, and ventilatory capacity is 1 ~ 5vvm.
8. bafillus natto liquor fermentation according to claim 1 prepares vitamin K2Method, it is characterised in that: institute
The relative energy density for stating Low Temperature Plasma Treating equipment is 25 ~ 60kW/l, is divided into 4-10mm, electrode diameter between electrode plate
40-120mm。
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